Transcriptome Analysis of Walnut (Juglans regia L.) Embryos Reveals Key Developmental Stages and Genes Involved in Lipid Biosynthesis and Polyunsaturated Fatty Acid Metabolism

Abstract

Walnut (Juglans regia L.) is a widely cultivated woody oilseed tree species, and its embryo is rich in polyunsaturated fatty acids. Thus far, the pathways and essential genes involved in oil biosynthesis in developing walnut embryos remain largely unclear. Our analyses revealed that a mature walnut embryo accumulated 69% oil, in which 71% were polyunsaturated fatty acids with 64% linoleic acid and 7% linolenic acid. RNA sequencing generated 39 384 unigenes in 24 cDNA libraries prepared from walnut embryos collected at 49, 63, 77, 91, 105, 119, 133, and 147 days after pollination (DAP). The principal components analysis (PCA) of samples and cluster analysis of differentially expressed genes (DEGs) showed that the total samples were divided into three main groups: 49 DAP, 63−119 DAP, and 133−147 DAP. We identified 108 unigenes associated with lipid biosynthesis, including 60 unigenes for fatty acid biosynthesis, 33 for triacylglycerol biosynthesis, 7 for oil bodies, and 8 for transcription factors. The expression levels of the genes encoding WRI1, ACCase, ACP, KASII, SAD, FAD2, FAD3, and PDAT were upregulated at 63− 119 DAP relative to the levels at 49 DAP. Additionally, the lipid biosynthesis in walnut embryos began to increase while oil contents increased from 15 to 69%. We identified eight SAD, three FAD2, one FAD3, one FAD5, one FAD6, and three FAD7/8 genes. In addition, SAD, FAD2, and FAD3 were highly abundantly expressed in the walnut embryo, and their FPKM values achieved were 834, 2205, and 9038, respectively. High expression levels of FAD2 and FAD3 may be the reason why walnuts are rich in polyunsaturated fatty acids. Subcellular localization confirmed that the JrFAD3 protein played a role in the endoplasmic reticulum rather than the plastid, suggesting that linolenic acid was mainly synthesized in the endoplasmic reticulum. Weighted gene coexpression network analysis (WGCNA) showed that ACP, ENO, VAMP727, and IDD14 were coexpressed with WRI1. Our study provides large-scale and comprehensive transcriptome data of walnut embryo development. These data lay the foundation for the metabolic engineering of walnuts to increase oil contents and modify fatty acid compositions.

Transcriptomic Analysis Reveals Key Genes Involved in Oil and Linoleic Acid Biosynthesis during Artemisia sphaerocephala Seed Development

“…This was basically consistent with the mechanism of C18:2 accumulation in G. hirsutum seeds [7]. The main FA of J. regia was also C18:2, the high expression of FAD3 and FAD2 was the main reason for the enrichment of polyunsaturated fatty acids, but the expression of FAD3 was higher than that of FAD2, and the mechanism of high C18:2 content remains to be further studied [5].…”

“…DGAT and PDAT are the key enzymes responsible for TAG biosynthesis in the acyl-CoA-dependent and acyl-CoA-independent pathways, respectively, and the relative contribution to TAG biosynthesis varied by species: PDAT had higher expression or higher correlation with seed oil content than DGAT in safflower (Carthamus tinctorius), J. regia, G. hirsutum, Torreya grandis and P. sibirica, indicating it may play a more important role in TAG biosynthesis [5,7,12,35,36]. Whereas in B. napus, DGAT was more important for the biosynthesis of TAG [18][19][20].…”

“…Seeds are important storage organs for vegetable oils, which are primarily stored as triacylglycerols (TAG), with variable content in different plant species. The oil content of walnut (Juglans regia) embryo, peanut (Arachis hypogaea), canola (Brassica napus), upland cotton (Gossypium hirsutum), and soybean (Glycine max) were found to be 69.1%, 58.2%, 42.7%, 35.2%, and 15.0~20.2%, respectively [3][4][5][6][7]. The accumulation of seed oil is a complex process, which primarily consists of two parts: FA de novo biosynthesis in plastids and TAG biosynthesis in endoplasmic reticulum (ER) [8,9].…”

“…The main UFA of Perilla (Perilla frutescens) and tree peony (Paeonia section Moutan) seed oil were C18:3, however, the formation of this trait was regulated by highly expressed FAD3 and FAD8, respectively [15,16]. G. hirsutum and walnut (Juglans regia) with C18:2 as the main FA, the high expression of FAD2 and the very low expression of FAD3 were the key reasons for the formation of G. hirsutum C18:2 [7], while in J. regia, the expression level of FAD3 was higher than that of FAD2, and the reason for the fact that the content of C18:3 was lower than that of C18:2 remains to be further studied [5]. The above results further indicate that the regulatory mechanism for the formation of the same FA trait varied from species to species.…”

Integration of miRNAs, Degradome, and Transcriptome Omics Uncovers a Complex Regulatory Network and Provides Insights Into Lipid and Fatty Acid Synthesis During Sesame Seed Development

“…Subsequently, the lipid biosynthetic pathway and key periods of FA biogenesis and associated proteins were deciphered in the developing endosperm of Cocos nucifera ( Reynolds et al, 2019 ) and tree peony seeds ( Yin et al, 2018 ), respectively. Similarly, regulatory networks of lipid and FA metabolism were revealed in Carya cathayensis Sarg ( Huang et al, 2016 , 2021 ) and oil palm ( Zheng et al, 2019 ) by transcriptomic and miRNA sequencing, respectively.…”